Laxative effect of ageratum conyzoides

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* Corresponding author: Vishweswar Rao.V
E-mail address: visu_rx@yahoo.com
Available online at www.ijrpp.com
Print ISSN: 2278 – 2648
Online ISSN: 2278 - 2656 IJRPP | Volume 2 | Issue 1 | 2013 Research article
Protective effect of an adenosine receptor agonist on neuropathic pain using
chemotherapy induced neuropathy in mice.
*
Vishweswar Rao.V, Sumadhuri Sreerama, Sowjanya Kumar Reddy.R, Santosh.M.
Vision College of Pharmaceutical sciences & Research, R.N.S.Colony, Boduppal, Ghatkesar (M),
R.R(Dist), Andhra Pradesh, India.
ABSTRACT
Neuropathy is defined as the pain condition that results from damage affecting peripheral nerves, posterior roots,
spinal cord or certain regions of brain. Increased neuronal excitability is thought to be the underlying mechanism for
all forms of painful neuropathies. The work was framed to study the adenosine based treatment for chemotherapy
induced neuropathy. Involvement of adenosine in nociception created an interest to work for chemotherapy induced
neuropathy. Tricyclic antidepressants (TCAs), though often the first choice in most patients causes sedation and
cardiovascular issues, Anticonvulsants, like the TCAs, are only partially effective in the majority of patients,
Opioids, though often prescribed for moderate to severe pain, are sometimes avoided because of their potential for
dependence and tolerance, scheduling issues and side effects. Hence in order to overcome these side effects we
made an attempt to develop a newer drug for the treatment. Mice were first treated with vincristine sulphate (100
µg/kg, i.v). A single intravenous dose of vincristine causes painful peripheral neuropathy. Then we administer drugs
for five days after inducing neuropathic pain. Baseline and 5 days after induction of neuropathic pain hyperalgesia
(mechanical) and allodynia (mechanical) of all groups were measured in order to confirm the development of
neuropathic pain. Hyperalgesia and allodynia of drug treated group was compared on 5th
day with vehicle treated
group in order to confirm the effectiveness of drug in neuropathy pain. Hence adenosine was found to be significant
against neuropathic pain in lower and also in higher dose. Hence adenosine may provide a better insight in the
development of the newer drug for neuropathic pain.
Key words: Neuropathy, Adenosine, Chemotherapy.
INTRODUCTION
Pain is an unpleasant sensory and emotional
experience associated with actual or potential tissue
damage1
. Pain is of two types nociceptive pain and
neuropathic pain. Nociceptive pain is the neural
processes of encoding and processing noxious
stimuli. It is the afferent activity produced in central
and peripheral nervous system by stimuli that have
the potential to damage tissue. Neuropathic pain is
defined as the pain condition that results from
damage affecting peripheral nerves, posterior roots,
spinal cord or certain regions of brain. Accumulation
of novel expression of sodium channels in periphery,
increased activity at glutamate receptor sub
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Pharmacology & Pharmacotherapeutics

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population, reduction of GABA inhibition and
changes in penetration of calcium in the cell are
reported as possible mechanism for the development
of neuropathic pain. Symptoms of neuropathic pain2
are 1. Stimulus dependent pain (can be shooting,
lancinating or burning) 2. Stimulus evoked pain is a
common component of peripheral nerve injury or
damage and has two key features. Hyperalgesia and
Allodynia.
Hyperalgesia is an increased pain response to a
suprathreshold noxious stimulus and is the result of
abnormal processing of nociceptor input. Allodynia is
the sensation of pain elicited by a non noxious
stimulus and can be produced by two ways 1. By the
action of low threshold myelinated A fibres on an
altered central nervous system. 2. By a reduction in
the threshold of nociceptor terminals in the periphery.
Stimulus evoked hyperalgesia are classified into
subgroups on the basis of modality mechanical,
thermal and chemical.
Mechanism involved in pain is complex and involve
both peripheral and central pathophysiological
phenomenon. Neuropathic pain is a severe
debilitating condition which affects approximately
six million people in US alone3
. Increased neuronal
excitability is thought to be the underlying
mechanism of all forms of painful neuropathies.
Therefore current pharmacotherapy of neuropathic
pain generally involves the use of drugs that either
reduces neuronal discharge or increases endogenous
antinociception system.
Sodium channel blockers, antiepileptic agents,
opioids, tricyclic antidepressents, gabapentin etc have
been employed to treat the painful symptoms of
different forms of neuropathy. Mainly adenosine play
a major role in the pathophysiology of neuropathic
pain for several reasons at the spinal cord, intrathecal
selective A1 adenosine receptor agonists inhibited C-
fiber evoked responses in the dorsal horn neurons in
rats4
. These results suggest a role of adenosine A1
receptor in the modulation of both acute and chronic
pain in autonomic nervous system is particularly
involved in neuropathic pain. Adenosine binds to
presynaptic A1 receptor and via a G1 protein leads to
a reduction in the cAMP concentration, in turn
leading to a decreased release of glutamate by
damaged nerve fibers which results in decrease in
neuropathic pain.
Hence in our present investigation Granisetron an
adenosine A1 receptor agonist (class of purinergic
receptors, G-protein coupled receptor with adenosine
as ligand) is used to know its protective effect on
neuropathic pain using chemotherapy (vincristine)
induced neuropathic pain.
MATERIALS AND METHODS
Drugs and chemicals
Sodium chloride was obtained from Sisco research
laboratories Pvt. Ltd, Mumbai. Gabapentin, was
purchased from Bava medicals, Pallavaram.
Vincristine and Granisetron were purchased from
Muthu pharmacy, Purasawalkam. All chemicals and
reagents used were of analytical grade.
Experimental animals
Adult male Swiss albino mice weighing 25-35 g were
used in the pharmacological Studies. The inbred
animals were taken from the animal house in Vel’s
College of Pharmacy, Pallavaram, Chennai-117. The
animals were housed in groups of 6 per cage. They
were maintained in well-ventilated room temperature
with relative humidity of 45-55% and natural 12h:
12h day-night cycle in propylene cages. They were
fed balanced rodent pellet diet from Poultry Research
Station, Nandanam, Chennai-35 and tap water
adlibitum throughout the experimental period. All the
experiments were carried out between 10:00 am to
5:00 pm. The animals were housed for one week,
prior to the experiments to acclimatize laboratory
temperature. Food, not water, was withdrawn 3 hrs
before and during experiment. The experiment
protocol was approved by the Institutional Animal
Ethics Committee IAEC Ref. No.
290/CPCSEA/2009-PH/PCOL-02.
Preparation of drug solution
Drug was dissolved in saline and administered to
animals through i.p. route.
Assessment of effectiveness of Granisetron for
neuropathic pain (Chemotherapy induced
neuropathic pain model)
Chemotherapy induced neuropathic pain was
performed following the method of5
Joseph et al.,
2003. Mice were first treated with vincristine
sulphate (100 µg/kg, i.v). A single intravenous dose
of vincristine causes painful peripheral neuropathy.

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Then we administer drugs for five days after inducing
neuropathic pain. Baseline and 5 days after induction
of neuropathic pain hyperalgesia (mechanical) and
allodynia (mechanical) of all groups were measured
in order to confirm the development of neuropathic
pain. Hyperalgesia and allodynia of drug treated
group was compared on 5th
day with vehicle treated
group in order to confirm the effectiveness of drug in
neuropathic pain
Experimental groups
The mice were divided into four groups consisting of
6 per each group
Group I -Vehicle- Saline (10 ml/kg, i.p.) for 5 days.
Group II- Granisetron (1 mg/kg, i.p) for 5 days.
Group III- Granisetron (10 mg/kg, i.p) for 5 days.
Group IV- Gabapentin (100 mg/kg, p.o) for 5 days.
Behavioral tests
Mechanical hyperalgesia6
Mechanical hyperalgesia was tested by using the pin
prick test. Animals were placed on the elevated grid;
a pin prick test was performed using a safety pin. The
lateral plantar surface of the right hind paw was
briefly stimulated at intensity sufficient to indent but
not penetrate the skin (pin prick test). The duration of
paw withdrawal was recorded, with an arbitrary
minimal time of 0.5 (sec) and a maximal cut off 15
(sec).
Mechanical Allodynia (Touch Evoked Tactile
Allodynia)7
Tactile allodynia was assessed by lightly stroking the
injured leg with a paintbrush. Allodynia response
was ranked as described by Minami et al. 1995.
0. No response.
1. Mild squeaking with attempts to move away from
the stroking probe.
2. Vigorous squeaking, biting the stroking probe and
strong efforts to escape from the stroking probe.
Assessment of effectiveness of Granisetron on
motor coordination
In order to assess whether the protection of
neuropathic pain of granisetron was via impaired
motor activity, motor coordination studies of
Granisetron was performed using the following
animal models.
Experimental groups
Mice were divided into three groups each group
having 6 animals.
Group I -Vehicle- 0.9% Saline (10 ml/kg, i.p.)
Group II- Granisetron (1 mg/kg, i.p)
Group III- Granisetron (10 mg/kg, i.p)
Behavioral tests
Rota rod Test8
Mice were placed on the rota-rod. Appropriate speed
of rotation was selected. Time taken by the animal to
fall off from the rotating rod was observed. A normal
(untreated) mouse generally falls off within 3 mins.
Locomotor Activity8
The locomotor activity can be easily measured using
actophotometer which operates on photoelectric cells
which are connected in a circuit with a counter.
When a beam of light falling on photocell is cut off
by the animal, a count is recorded. Mice were
individually placed in the activity cage for 10 mins
and the scores were recorded.
Statistical analysis
The data are represented as mean ±S.E.M and
statistical significance between groups were analysed
by means of student paired t-test, ANOVA followed
by Dunnet’s t-test, as applicable. P<0.05 implies
significance. All the statistical analysis was carried
out using graph pad prism 5.0 version software.
RESULTS
Vincristine induced neuropathic pain model
Development of signs of neuropathic pain
All the vincristine treated animals developed
qualitative signs indicative of neuropathic pain. This
was clearly present on the 5th
day after administration
of vincristine at a dose of 100 µg/kg, i.v. Table 1,
Table 2 showed vincristine treated group paw
withdrawal latency was reduced on 5th
day in
comparison to basal reading of the same animals. The
paw withdrawal latency of the animal was decreased
and allodynia score on 5th
day was significantly
increased in comparison to baseline measurements
indicating the development of hyperalgesia and
allodynia.

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Effect of Granisetron on Mechanical
Hyperalgesia
In the vehicle treated animals, the response duration
decreased at 5th
day in comparison to baseline
measurements (Table 1) from 14.33±0.33 to
4.00±0.36**
on the 5th
day. Whereas the other two
groups treated with granisetron at a dose of 1mg/kg
and 10 mg/kg from 14.33±0.33 to 7.23±0.42*and
9.26±0.28** the response duration was significantly
increased at both the dose levels. Thus
administration of Granisetron at both dose levels
shows the increased the paw withdrawal latency in
comparison to the vehicle treated group (Figure 1).
The results are dose dependent and comparable with
that of standard drug gabapentin.
Table 1: Development of mechanical hyperalgesia in chemotherapy induced
neuropathic pain model.
Statistical significance test was done by paired t test.
Values are mean± S.E.M of 6 animals per group.
Comparison was made between base line Vs 5th
day of the vehicle treated groups.
**P<0.01
Effect of Granisetron on Mechanical
allodynia
The baseline paw withdrawal frequencies (0.5 (sec))
determined by mechanical stimulation with paint
brush was enhanced at 5th day in vehicle treated mice
compared to baseline responses of the same animals.
Granisetron treated group at a dose level of 1mg/kg
and 10mg/kg decreased the allodynia score from the
value of 3.16±0.16** to 2.50±0.14** for 1mg/kg and
2.20±0.18** for 10mg/kg. The values are statistically
significant and the results were comparable with that
of standard drug gabapentin 100 mg/kg, p.o (Figure
2).
Table 2: Development of mechanical allodynia in chemotherapy induced
neuropathic pain model
Statistical significance test was done by paired t test.
Values are mean± S.E.M of 6 animals per group.
Comparison was made between base line Vs 5th day of the vehicle treated groups.
**P<0.01
S.No Treatment Paw withdrawal latency(sec)
Baseline 5th
day
1 Vehicle-Saline(10ml/kg,
i.p) 14.33±0.33 4.00±0.36**
S.No Treatment Allodynia Score
Baseline 5th
day
1 Vehicle-Saline(10 ml/kg,
i.p) 0.00±0.00 3.16±0.16**

5. Vishweswar Rao.V et al / Int. J. of Res.
Figure 1: Effect of Granisetron on mechanical hyperalgesia in chemotherapy induced
Statistical significance test was done by one way ANOVA followed by Dunnet’s
Values are mean± S.E.M of 6 animals per group.
Comparison was made between vehicle Vs 5th day of the all groups.
***P<0.001
Figure 2: Effect of Granisetron on mechanica
Statistical significance test was done by one way ANOVA followed by Dunnet’s
Values are mean± S.E.M of 6 animals per group.
Comparison was made between vehicle Vs 5
*P<0.05
**P<0.01
DISCUSSION
The mice chemotherapy-induced neuropathic pain
model utilized in the present study is one of many;
diverse animal models that have been used to
investigate the pharmacological attenuation
neuropathic pain9
. Rodent models of neuropathy have
been developed for three of the most widely used and
most neurotoxic cancer drugs – cisplatin, paclitaxel
and vincristine. These models have all been used to
determine the efficacy of neuroprotective a
and to study the mechanisms of sensory dysfunction
that underlie neuropathy11
. However, the vincristine
0
20
Vehicle
pawwithdrawal
latency(sec)
Mechanical hyperalgesia
0
2
4
Vehicle
AllodyniaScore
Mechanical allodynia
et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(1) 2013 [2
www.ijrpp.com
Effect of Granisetron on mechanical hyperalgesia in chemotherapy induced
neuropathic pain model
Statistical significance test was done by one way ANOVA followed by Dunnet’s t test.
Values are mean± S.E.M of 6 animals per group.
Comparison was made between vehicle Vs 5th day of the all groups.
Figure 2: Effect of Granisetron on mechanical allodynia in chemotherapy induced
neuropathic pain model
Statistical significance test was done by one way ANOVA followed by Dunnet’s t test
Values are mean± S.E.M of 6 animals per group.
Comparison was made between vehicle Vs 5th
day of the all groups.
induced neuropathic pain
model utilized in the present study is one of many;
diverse animal models that have been used to
investigate the pharmacological attenuation of
. Rodent models of neuropathy have
been developed for three of the most widely used and
cisplatin, paclitaxel
and vincristine. These models have all been used to
determine the efficacy of neuroprotective agents10
and to study the mechanisms of sensory dysfunction
. However, the vincristine
and paclitaxel models have several advantages over
cisplatin models. General health effects are an issue
for cisplatin models. Low cumulative do
paclitaxel or vincristine produce quantifiable changes
in sensory thresholds without decreasing motor
function or significantly impairing general health
To date, no drug or drug class is considered to be
both a ‘safe and effective analgesi
of chemotherapy induced pain. Tricyclic
antidepressants (TCAs), though often the first choice
in most patients, have significant side effects
including sedation and various cardiovascular issues,
and they often require several days of t
*** ***
Granisetron 1 mg/kg Granisetron 10 mg/kg Gabapentin 100 mg/kg
5th day
Mechanical hyperalgesia
* * **
Granisetron 1 mg/kg Granisetron 10 mg/kg Gabapentin 100 mg/kg
5th day
Mechanical allodynia
283
[279-285]
Effect of Granisetron on mechanical hyperalgesia in chemotherapy induced
l allodynia in chemotherapy induced
and paclitaxel models have several advantages over
cisplatin models. General health effects are an issue
for cisplatin models. Low cumulative doses of either
paclitaxel or vincristine produce quantifiable changes
in sensory thresholds without decreasing motor
function or significantly impairing general health12
.
To date, no drug or drug class is considered to be
both a ‘safe and effective analgesic’ in the treatment
of chemotherapy induced pain. Tricyclic
antidepressants (TCAs), though often the first choice
in most patients, have significant side effects
including sedation and various cardiovascular issues,
and they often require several days of treatment prior
***
Gabapentin 100 mg/kg
**
Gabapentin 100 mg/kg

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to producing positive effects13, 14
. Anticonvulsants,
like the TCAs, are only partially effective in the
majority of patients suffering from chemotherapy
induced pain14, 15
. Opioids, though often prescribed
for moderate to severe pain, are sometimes avoided
because of their potential for dependence and
tolerance, scheduling issues and side effects16
. Other
therapeutic agents are prescribed for the treatment of
cancer pain, in general. For example, non-steroidal
anti-inflammatory drugs (NSAID) have been reported
as having some analgesic efficacy in cancer
pain2,17,18,19,20
. However, prolonged NSAID usage can
be associated with significant toxicity, including that
to the gastrointestinal system and liver17
. Epidural
clonidine, an α2-adrenoceptor agonist, is indicated (in
combination with opioids) for the treatment of severe
cancer pain, however, profound hypotension can be a
side effect. Hence in order to overcome these side
effects we made an attempt to study the vincristine
induced neuropathy. Vincristine is basically a mitotic
inhibitor. They bind to tubulin, prevents its
polymerization and assembly into microtubule,
causing disruption of mitotic spindle thereby
interferering with cytoskeletal function. Vincristine
also inhibits cellular activity of microtubules like
axonal transport in neurons. Thus they disrupt the
normal axonal transport and lead to change in
excitability of neurons thus producing chronic pain.
A single intravenous dose of vincristine (50, 100, or
200 mg kg-1
) causes a painful peripheral neuropathy
in rats verified by mechanical hyperalgesia and
mechanical allodynia. A single administration of
vincristine is well tolerated at all of the doses studied
(50, 100 or 200 mg /kg) Rats treated with vincristine
gain weight at a slower rate than control rats but there
are no marked differences in the general appearance
of control versus vincristine-treated rats. There are
also no motor deficits in rats treated with a single
dose of vincristine. Models in which neuropathy is
induced by multiple doses of vincristine produce a
rapid-onset painful neuropathy but cumulative doses
more than 500 mg/kg also produce moderate to
severe effects on general health and motor deficits in
most studies21
. At cumulative doses more than 750
mg/kg vincristine causes significant mortality22
.
So in our present study we have studied the role of
Granisetron against the single dose vincristine
induced neuropathy pain. Results showed that
Granisetron at lower and higher dose level of 5 days
administration attenuated mechanical and mechanical
allodynia.
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